Tumor-derived TGF-β1 promoted STAT3 activation by inhibiting Myo1f expression. (A) KEGG pathway. Blue star indicates phospho-STAT3 signaling. (B) Tumor collection at day 27 (N = 5). (C) Endogenous IP (co-IP) blot with BM neutrophils from WT mice, where IP was performed for TRIM21, and then precipitates were immunoblotted (IB) with an anti-MYO1F antibody. (D) Exogenous co-IP blot with transfection of Flag-tagged TRIM21 and His-tagged MYO1F in 293T, where IP was performed for Flag, and then IB with an anti-His antibody. (E) Cultured WT neutrophils was treated with TGF-β1; MYO1F protein levels were analyzed 72 h after treatment by immunoblotting. (F) Top: FACS analyses of cytoplasmic TGF-β1 in different cell lines and neutrophils. Bottom: ELISA analysis of TGF-β1 from supernatant of cell lines. (G) Gate strategy of intratumoral immune cells from WT B16F10 tumor model. (H and I) FACS analyses of cytoplasmic TGF-β1 in different tumor-infiltrated immune cells and statistics of TGF-β1 MFI level. (J) Statistics of immune cells in total tumor cells as described in H. (K and L) FACS analyses of cytoplasmic TGF-β1 in B16F10 cells transfected with sgCtrl or sgTgfb1; ELISA analysis of TGF-β1 from supernatant of B16F10 cells (N = 3). (M) Counts of total CD11b+ Ly6G+ neutrophils in one tibia by FACS (N = 5). (N) Detecting STAT3 phosphorylation level of cultured neutrophils with overexpression of Flag-tagged MYO1F through western blotting, ratio of phosphor-STAT3 to total STAT3 was shown in the middle. (O) Detecting STAT3 phosphorylation level of cultured neutrophils with knockdown of MYO1F by siMyo1f, ratio of phospho-STAT3 to total STAT3 was shown in the middle. (P) SPI1-binding sites on Myo1f predicted by JASPAR. (Q)Myo1f mRNA levels were detected by qPCR from sorted immune cells (N = 3). (R) Transcription-related sites and SPI1-binding sites predicted by Softberry. Data in B–O and Q represent one experiment of three independent repeats. Data are presented as mean ± SD. P values were analyzed by one-way ANOVA test (M and Q); two-tailed unpaired Student’s t test (K and L), *P < 0.05, **P < 0.01, and ***P < 0.001. ns, no significance. Neu, neutrophil. Source data are available for this figure: SourceData FS5.
Figure S5.

Tumor-derived TGF-β1 promoted STAT3 activation by inhibiting Myo1f expression . (A) KEGG pathway. Blue star indicates phospho-STAT3 signaling. (B) Tumor collection at day 27 (N = 5). (C) Endogenous IP (co-IP) blot with BM neutrophils from WT mice, where IP was performed for TRIM21, and then precipitates were immunoblotted (IB) with an anti-MYO1F antibody. (D) Exogenous co-IP blot with transfection of Flag-tagged TRIM21 and His-tagged MYO1F in 293T, where IP was performed for Flag, and then IB with an anti-His antibody. (E) Cultured WT neutrophils was treated with TGF-β1; MYO1F protein levels were analyzed 72 h after treatment by immunoblotting. (F) Top: FACS analyses of cytoplasmic TGF-β1 in different cell lines and neutrophils. Bottom: ELISA analysis of TGF-β1 from supernatant of cell lines. (G) Gate strategy of intratumoral immune cells from WT B16F10 tumor model. (H and I) FACS analyses of cytoplasmic TGF-β1 in different tumor-infiltrated immune cells and statistics of TGF-β1 MFI level. (J) Statistics of immune cells in total tumor cells as described in H. (K and L) FACS analyses of cytoplasmic TGF-β1 in B16F10 cells transfected with sgCtrl or sgTgfb1; ELISA analysis of TGF-β1 from supernatant of B16F10 cells (N = 3). (M) Counts of total CD11b+ Ly6G+ neutrophils in one tibia by FACS (N = 5). (N) Detecting STAT3 phosphorylation level of cultured neutrophils with overexpression of Flag-tagged MYO1F through western blotting, ratio of phosphor-STAT3 to total STAT3 was shown in the middle. (O) Detecting STAT3 phosphorylation level of cultured neutrophils with knockdown of MYO1F by siMyo1f, ratio of phospho-STAT3 to total STAT3 was shown in the middle. (P) SPI1-binding sites on Myo1f predicted by JASPAR. (Q)Myo1f mRNA levels were detected by qPCR from sorted immune cells (N = 3). (R) Transcription-related sites and SPI1-binding sites predicted by Softberry. Data in B–O and Q represent one experiment of three independent repeats. Data are presented as mean ± SD. P values were analyzed by one-way ANOVA test (M and Q); two-tailed unpaired Student’s t test (K and L), *P < 0.05, **P < 0.01, and ***P < 0.001. ns, no significance. Neu, neutrophil. Source data are available for this figure: SourceData FS5.

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